Oral fluids (OFs) contain a diverse array of extracellular vesicles (EVs) that hold promise as a source of diagnostic information. Developing EV-based diagnostics using OFs requires an understanding of the physicochemical properties and heterogeneity of these EVs. This review explores strategies for differentiating EVs in OFs, including differential centrifugation, density gradient centrifugation, and a novel method based on sedimentation patterns. These techniques have revealed distinct subpopulations of EVs in OFs, each associated with specific biological functions and potential diagnostic utility. However, the complexity of EVs in OFs presents challenges, and a comprehensive understanding of their biogenesis and composition is still emerging. Future research should focus on refining EV isolation methods and exploring the diagnostic potential of both EV and non-EV particles in OFs.